Schoppink P J, De Jong M, Berden J A, Grivell L A
Laboratory of Biochemistry, University of Amsterdam, The Netherlands.
Eur J Biochem. 1989 May 15;181(3):681-7. doi: 10.1111/j.1432-1033.1989.tb14777.x.
Inactivation of the gene encoding the 11-kDa subunit VIII of yeast ubiquinol:cytochrome c oxidoreductase leads to an inactive complex, which lacks detectable cytochrome b [Maarse, A. C., De Haan, M., Schoppink, P. J., Berden, J. A. and Grivell, L. A. (1988) Eur. J. Biochem. 172, 179-184] and in which the steady-state levels of the Fe-S protein and the 14-kDa subunit VII are severely reduced. When the 11-kDao mutant is transformed with a gene encoding a protein consisting of the 11-kDa protein minus its last 11 amino acids and fused to a 7-amino-acid sequence encoded by a stop oligonucleotide, the complex is assembled normally. Enzyme activity is similar to that of the wild type, as is also the sensitivity of the complex to antimycin and myxothiazol. Transformation of the mutant with a gene encoding a protein consisting of the 11-kDa protein lacking the last 43 amino acids (i.e. almost half the protein) and fused to the same 7-amino-acid sequence as above, gives partial restoration of the complex. The Fe-S protein and the 14-kDa subunit VII still exhibit low steady-state levels, but cytochrome b is present again, albeit at a strongly reduced level. Electron transport activity is also partially restored and correlates with the level of cytochrome b indicating that the turnover number of the complex is similar to that of wild-type complex III. These findings demonstrate the important role played by the 11-kDa protein in the stabilization of cytochrome b. They also imply that at least the C-terminal half of the 11-kDa protein is not part of an ubiquinol-binding site. Moreover, since the deletion has no effect on the sensitivity of the complex to myxothiazol and antimycin, at least this part of the protein is probably not involved in binding of these inhibitors.
细胞色素c氧化还原酶11 kDa亚基VIII编码基因的失活会导致复合物失活，该复合物缺乏可检测到的细胞色素b [马尔塞，A.C.，德哈恩，M.，朔平克，P.J.，贝登，J.A.和格里维尔，L.A.（1988年）《欧洲生物化学杂志》172卷，179 - 184页]，并且其中铁硫蛋白和14 kDa亚基VII的稳态水平严重降低。当用一个编码由11 kDa蛋白减去其最后11个氨基酸并与由终止寡核苷酸编码的7个氨基酸序列融合而成的蛋白质的基因转化11 kDa突变体时，复合物能正常组装。酶活性与野生型相似，复合物对抗霉素和粘噻唑的敏感性也与野生型相似。用一个编码由11 kDa蛋白缺失最后43个氨基酸（即几乎该蛋白的一半）并与上述相同的7个氨基酸序列融合而成的蛋白质的基因转化该突变体，可使复合物部分恢复。铁硫蛋白和14 kDa亚基VII的稳态水平仍然较低，但细胞色素b再次出现，尽管水平大幅降低。电子传递活性也部分恢复，并且与细胞色素b的水平相关，这表明该复合物的周转数与野生型复合物III相似。这些发现证明了11 kDa蛋白在细胞色素b稳定中所起的重要作用。它们还意味着至少11 kDa蛋白的C末端一半不是泛醇结合位点的一部分。此外，由于该缺失对复合物对粘噻唑和抗霉素的敏感性没有影响，至少该蛋白的这部分可能不参与这些抑制剂的结合。
